Negative cooperativity between folinic acid and coenzyme in their binding to Lactobacillus casei dihydrofolate reductase.
The binding of folinic acid (5-formyl-5,6,7,8-tetrahydrofolate) to Lactobacillus casei dihydrofolate reductase has been measured. The natural 6S, alpha S diastereoisomer has a binding constant of 1.3 (+/- 0.6) X 10(8) M-1 at pH 6.0, 25 degrees C; the 6R, alpha S diastereoisomer binds approximately 10(4)-fold more weakly. The natural diastereoisomer of folinic acid binds negatively cooperatively with the coenzymes NADP+ and NADPH, binding 3 times more weakly in the presence of NADP+ and 600 times more weakly in the presence of NADPH than to the enzyme alone. Negative cooperativity has been unequivocally distinguished from competition by measurements of coenzyme binding as a function of folinic acid concentration, of the effects of folinic acid on the 1H and 31P chemical shifts of the bound coenzyme, and of the effects of folinic acid on the coenzyme dissociation rate constant. The latter experiments also give evidence for the coexistence of two slowly interconverting conformational forms of the ternary enzyme-coenzyme-folinic acid complex. Small changes in structure of the oxidized coenzymes have substantial effects on the cooperativity with folinic acid, with the thionicotinamide analogue showing positive rather than negative cooperativity. The changes in environment of the bound coenzyme produced by folinic acid, as revealed by 1H and 31P NMR, demonstrate clearly that the negative cooperativity shown by NADP+ and NADPH, respectively, arises by two structurally distinct mechanisms.
D002151:Calorimetry; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D002955:Leucovorin / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D009249:NADP; D013762:Tetrahydrofolate Dehydrogenase; D002955:Leucovorin